Evanescent field-based sensors are fast becoming a technology of choice for accurate label-free detection of a biological, biochemical, or chemical substance (e.g., cells, spores, biological or drug molecules, or chemical compounds). This technology typically involves using a grating-coupled waveguide (GCW) to sense a concentration change, surface adsorption, reaction, or the mere presence of a biological or chemical substance at the waveguide surface. These detectable events are manifested as a change in the effective refractive index of a waveguide mode that partially or completely overlaps the sensing region (waveguide substrate). To generate the evanescent or optical field, an optical interrogation system uses optical elements, such as a grating or prism, to couple a light beam from a light source in and out of an optical mode in the waveguide of the GCW sensor. The optical interrogation system also includes a detector that receives the light beam coupled out from the waveguide. The angle or wavelength of the emitted light beam is analyzed to determine the effective refractive index of the waveguide. Changes in the angle or wavelength of the probe light, for example, indicate changes of the waveguide effective index that result from activity at the sensor surface.
In determining the effective refractive index of the GCW sensor, the principles of optical-physics dictate that the light beam received by the detector had interacted with the waveguide under a resonant condition, where the wavevectors of a diffraction grating, incoming light beam, and guided mode all sum to zero, thereby allowing one to probe the effective index of the mode, which changes together with the surface index. This resonant condition occurs only for a specific wavelength and angle of the incoming light, and changes in this angle or wavelength correspond to changes in the effective refractive index of the waveguide caused by the concentration changes, surface adsorption, or reactions of biological or chemical substances in the sensing region of the GCW sensor. Thus, the optical interrogation system is used to sense a change in the effective index of the GCW sensor which enables one to determine whether or not a substance of interest is located within the sensing region of the GCW sensor.
For this technology to be viable, one must have an optical interrogation system and in particular a detector capable of accurately monitoring the resonant angle, the wavelength, or both. In particular, the optical interrogation system must emit a light beam that interacts with the GCW sensor, and must in turn receive the light beam coupled-out of the GCW sensor and process that light beam to detect in real time any changes in the resonant angle and/or wavelength of the light beam. While there are many approaches for accomplishing these tasks, each has unique challenges associated with implementation, since the light beam output from the GCW sensor may be relatively weak and the presence of multiple sources of noise can degrade the light beam, especially in high-throughput screening applications.
Evanescent- or optical-field sensors have demonstrated both high sensitivity and an ability to detect binding reactions of as little as about 250 Da molecular weight (e.g., biotin binding to streptavidin). In recent years, the biological, pharmaceutical, and other research communities have begun to recognize that optical field-based sensors can be useful, high-throughput research tools to measure a variety of biological or biochemical functions. GCW sensors are particularly attractive for use in high-throughput screening applications, where the absence of fluorescent tags and the possibility of reduced false-negatives would provide a large cost advantage. For this reason, microtiter well plates, also known as microplates, have caught the attention of researchers as a promising platform for such sensors, where 96 or 384 individual wells provide the high-throughput access demanded by the industry. When applied in the context of a microplate, the waveguide and diffraction grating of the GCW sensor are preferably located in the bottom of each well (e.g., the diffraction grating may be stamped or otherwise molded into the well bottom, and the waveguide is subsequently applied on top of the diffraction grating). The wells themselves are typically composed of an optically transparent, low-birefringence, preferably low-cost plastic that is typically about several hundreds of microns to about a few millimeters thick. Plates fabricated on glass substrates also are suitable for these applications.
In the context of a high-throughput screening application, microtiter well plates will be handled by various types of robotic instruments. During the course of robotic manipulations fluids will be added and removed from individual wells, assay protocols may require incubation periods; hence, the microplate will likely as a consequence be inserted and reinserted into the sensor or detection device more than once during a single assay measurement cycle. Since the resonance condition of the GCW sensor is critically dependent on the angle of the light striking the microplate, repositioning of the plate in the detection device will manifest itself (at least in part) in the form of angular noise in the sensor instrument. This environmental perturbation can in fact be much larger than and overwhelm the sought-after response of the GCW sensor to true biological or chemical changes in the sensing region. Thus, this problem can work at cross-purposes with a sensor that is designed purposefully to enhance or maximize sensitivity to its biochemical environment. In other words, the sensor's extra-sensitivity can exacerbate environmental background noise. The simultaneous desire for an extremely responsive sensor with high biochemical sensitivity and need for low susceptibility to environmental responses place unique constraints on the system designer. The present invention can balance these two competing requirements, and the optical interrogation system, GCW sensor, and method of the present invention successfully address and satisfy this difficult problem.